Imaging Cellular Proliferation in Prostate Cancer with Positron Emission Tomography.

Prostate cancer remains a major public health problem worldwide. Imaging plays an important role in the assessment of disease at all its clinical phases, including staging, restaging after definitive therapy, evaluation of therapy response, and prognostication. Positron emission tomography with a number of biologically targeted radiotracers has been demonstrated to have potential diagnostic and prognostic utility in the various clinical phases of this prevalent disease. Given the remarkable biological heterogeneity of prostate cancer, one major unmet clinical need that remains is the non-invasive imaging-based characterization of prostate tumors. Accurate tumor characterization allows for image-targeted biopsy and focal therapy as well as facilitates objective assessment of therapy effect. PET in conjunction with radiotracers that track the thymidine salvage pathway of DNA synthesis may be helpful to fulfill this necessity. We review briefly the preclinical and pilot clinical experience with the two major cellular proliferation radiotracers, [(18)F]-3'-deoxy-3'-fluorothymidine and [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil in prostate cancer.


Introduction
An important unmet clinical need in the imaging evaluation of prostate cancer is image-based characterization of tumor, which can facilitate clinical decision-making and patient management. Prostate cancer has a wide spectrum of biological behavior that ranges from indolent to aggressive. While indolent tumors may be managed with active surveillance, aggressive tumors will need early definitive treatment for improved patient outcome.
Imaging cellular proliferation may provide valuable diagnostic information about the rate of tumor growth and an opportunity for objective assessment of response to treatment (3)(4)(5). PET in conjunction with radiotracers that track the thymidine salvage pathway of DNA synthesis has been studied relatively extensively for noninvasive imaging-based assessment of cellular proliferation in cancer (6,7). Although 11 C-thymidine was an early contender, but major limitations were encountered primarily in relation to rapid catabolism of thymidine (8)(9)(10)(11). Further research resulted in the development of analogs that were resistant to catabolism and can be labeled with the longer half-life 18 F (110 min) which in turn facilitates regional distribution of the tracer without the need for an on-site cyclotron. Here we briefly highlight the experience with two of these radiotracers that have been employed in preclinical and pilot clinical studies in prostate cancer, [ 18 F]-3'-deoxy-3'-fluorothymidine and [ 18 F]-2'-fluoro-5-methyl-1beta-D-arabinofuranosyluracil ( Figure 1).
In another preclinical micro PET study, a significant decline in 18 F-FLT uptake was noted in the 22Rv1 hormone-refractory prostate tumors implanted in athymic mice after treatment with docetaxel (19). Interestingly, in this study, changes in prostate-specific antigen concentration in the cell medium and 18 F-FDG uptake in response to treatment were minimal. The authors concluded that 18 F-FLT is a promising tracer for early assessment of anticancer therapy with docetaxel in patient with hormone refractory prostate cancer. Therefore, while it appears that 18 F-FLT may be helpful in the evaluation of treatment response in prostate cancer, the exact utility of 18 F-FLT in this context remains unsettled, especially given the fact that there is high physiologic localization of the radiotracer in the normal bone marrow that is the most common site for prostate tumor metastases.

[ 1 8 F ] -2 ' -f l u o r o -5 -m e t h y l -1 -b e t a -Darabinofuranosyluracil
This thymidine analog is phosphorylated by thymidine kinase and incorporated in the DNA. The unlabeled compound (abbreviated as FMAU) was originally of clinical interest as an anticancer and an antiviral drug when used in pharmacological dose (20). Tehrani et al. showed that this thymidine analog is preferentially phosphorylated by the mitochondrial thymidine kinase 2 (TK2) in comparison to the cytosolic TK1 (21). In tracer doses, this agent can be labeled with 11 C or 18 F and as such are useful for imaging DNA synthesis and tumor proliferation (22)(23)(24)(25)(26). It has also been used for imaging reporter gene expression using the herpes simplex virus type 1 thymidine kinase (HSV-tk1) system (27)(28)(29)(30). Recently, an automated cGMP-compliant radiosynthesis of FMAU has been described (31).
Pharmacokinetic studies have shown that 14 C-FMAU behaves very similar to the pyrimidine nucleoside, thymidine, with respect to cellular uptake velocity, saturability of cellular incorporation, and intracellular metabolite pools and is reflective of tumor cell division (32). A recent report from our laboratory at the University of Southern California showed that 11 C-FMAU uptake in a dog brain tumor model correlated with tumor growth rate and could be well described by a three-compartment kinetic model (33). The adequacy of three-compartment model has also been shown for 18 F-FMAU (34). One study comparing the L-isomer with the D-isomer showed higher accumulation of the D-isomer in both fast growing H441 (byafactorofabout7.74), and slow growing H3255 (by a factor of about 3.37) human lung cancer cell lines (35). Of note, these values were significantly higher than those for the L-isomer 18 F-FMAU and 18 F-FLT.
Initial imaging-based biodistribution of 18 F-FMAU in normal dogs have shown that 18 F-FMAU is resistant to degradation, and is selectively retained in DNA (36). 18 F-FMAU shows little accumulation in bone (a common site for metastasis from prostate cancer) that renders it a potentially ideal PET radiotracer for imaging DNA synthesis in prostate cancer (37) (Figure 3). Recently our laboratory showed that there may be an association between androgen signaling and thymidine metabolism and that 18 F-FMAU PET may be useful in prostate tumor characterization (38). One possibility may be the androgen control of mitochondrial function that may include TK2 enzymatic activity (39).
A pilot observational study of 18 F-FMAU PET in three men with prostate cancer confirmed tumor retention of 18 F-FMAU in local prostate recurrence, and in metastatic lesions with barely visible activity in the urinary bladder and the normal bone (in prostate recurrence: tumor-to-background pelvis activity ratio of 2.3-6.3; in bone metastasis: tumor-to-background normal bone activity ratio of 2.4-3.1) (40). Moreover, on average, 95% of the blood activity cleared within 10 minutes post 18 F-FMAU administration, and about 70% of the activity in the urine was intact 18 F-FMAU at 60 minutes post injection. We have also recently initiated a pilot study to assess the potential utility of 18 F-FMAU in image-targeted biopsy using sophisticated software-based fusion of PET, transrectal ultrasound and magnetic resonance imaging of the prostate gland. Such hybrid imaging methodology may allow for improved localization and characterization of tumors for targeted biopsy and focal therapy. Additional applications may include the use of 18 F-FMAU in the assessment of treatment response, and prognosis in men with  Although other substituted 2'-[ 18 F]fluro-2'deoxy-arabinofuranosyluracil derivatives such as 2'-deoxy-2'-[ 18 F]fluoro-5-bromo-1-beta-Darabinofuranosyluracil ( 18 F-FBAU), 2'-deoxy-2'[ 18 F]fluro-5-chloro-1-beta-D-arabinofuranosyluracil ( 18 F-FCAU), 2'-deoxy-2'-18 F-fluoro-5-fluoro-1-beta-D-arabinofuranosyluracil ( 18 F-FFAU), and others have also been synthesized, but their exact clinical utility and potential competitive advantage over 18 F-FLT and 18 F-FMAU will need further exploration (41,42).

Conclusions
Imaging cellular proliferation in prostate cancer allows for personalized precision care in men with prostate cancer.
Imaging-based tumor characterization allows for improved clinical decision-making through management stratification that may lead to enhanced patient outcome, decreased adverse events and lower cost of care. Additional prospective investigations of imaging cellular proliferation in prostate cancer are warranted.